Emerging Chagas Disease: Trophic Network and Cycle of Transmission of Trypanosoma cruzi from Palm...

100Emerging Infectious Diseases Vol. 7, No. 1, January–February 2001

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The tropical moist broadleaf forests of LatinAmerica are an important region for conservationof biodiversity (1). In the Amazon Basin (area8,214,284 km2), blocks of original habitat are stillintact, while some ecoregions1 are almostcompletely converted or degraded, allowingmajor components of biodiversity to steadilyerode (1,2). Populations of several endangeredwildlife species have declined, and humanhabitat and land use are considered a threat tomost native species and communities.

Palm trees propagate in ecoregions of theAmazon Basin ecosystem amid other vegetationor in enormous palm forests. Of approximately2,800 palm species worldwide, 387 (13.8%) arenative to the basin (3,4). Palm trees have been

used to study the evolution of biological diversityand are excellent markers of ecologic fitness inthe Amazon Basin (2,4). In addition, these treesmay play an important role in the forestecosystem. Palm trees produce 15 tons of dryorganic material per hectare per year (threefoldmore than other species of trees) and recuperatemore rapidly after fire than other forest species.A single native palm tree may serve as shelterand food for diverse fauna (wild mammals,snakes, scorpions, spiders, amphibians, andmany species of insects). Palm trees are also animportant economic resource for residents of theAmazon region, who collect and sell palm roots,stipe, leaves, fruits, seeds, heart of palm, andinflorescences (4,5). The babassu palm (Attalaeaphalerata) reaches an average density of 200trees per hectare in the state of Maranhão, butlower densities were reported in the states ofPiaui, Goiás-Tocantins, and Mato Grosso (5), wherebabassu trees number an estimated 11 x 108.

Emerging Chagas Disease:Trophic Network and Cycle of

Transmission of Trypanosoma cruzifrom Palm Trees in the Amazon

Antonio R.L. Teixeira,* Pedro Sadi Monteiro,* José M. Rebelo,†Enrique R. Argañaraz,* Daniela Vieira,* Liana Lauria-Pires,*Rubens Nascimento,* Cássia A. Vexenat,* Antonio R. Silva,†

Steven K. Ault,‡ and Jackson M. Costa‡*University of Brasília, †Federal University of Maranhão, and ‡Pan-American

Health Organization/World Health Organization, Brasília, Brazil

Address for correspondence: Antonio R.L. Teixeira, Chagas’Disease Multidisciplinary Research Laboratory, Faculty ofMedicine, University of Brasília. P.O. Box 04536. Postcode70.919.970, Brasília, DF, Brazil; fax: 55+61 273-4645; e-mail:[email protected].

A trophic network involving molds, invertebrates, and vertebrates, ancestrallyadapted to the palm tree (Attalaea phalerata) microhabitat, maintains enzooticTrypanosoma cruzi infections in the Amazonian county Paço do Lumiar, state ofMaranhão, Brazil. We assessed seropositivity for T. cruzi infections in the humanpopulation of the county, searched in palm trees for the triatomines that harbor theseinfections, and gathered demographic, environmental, and socioeconomic data.Rhodnius pictipes and R. neglectus in palm-tree frond clefts or in houses were infectedwith T. cruzi (57% and 41%, respectively). Human blood was found in 6.8% of R. pictipesin houses, and 9 of 10 wild Didelphis marsupialis had virulent T. cruzi infections.Increasing human population density, rain forest deforestation, and human predation oflocal fauna are risk factors for human T. cruzi infections.

1In this study, a major ecosystem is defined as a set of ecoregions of comparable dynamics, response characteristics to disturbance,species diversity, and conservation needs. An ecoregion is a geographically distinct set of natural communities with similar species,ecologic dynamics, environmental conditions, and ecologic interactions critical for long-term persistence (1).

101Vol. 7, No. 1, January–February 2001 Emerging Infectious Diseases

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Not much attention has been given to humanhealth conditions in this ecosystem (6), probablybecause pathologic conditions in tropical broad-leaf forests are difficult to quantify in theseisolated, often impoverished communities. Nine-teen sylvatic species of triatomines were identifiedin the Amazon Basin (7-10), six in associationwith palm tree microhabitats (Table 1). Eleven of

these species were infected with Trypanosomacruzi or T. cruzi-like flagellates (17,18). None ofthese triatomine species, with the possibleexception of T. rubrofasciata, have adapted tohuman habitats in the Amazon Basin (19). Since1924, when T. cruzi infection in wild squirrelmonkeys (Crisotrix sciureus) was described (20),sporadic human T. cruzi infections have beenreported in the basin (21). However, this enzooticprotozoan infection received attention only after1969, when acute cases of human Chagas diseasewere described in Belém, State of Pará, Brazil(22,23). Further evidence shows that T. cruziinfections are endemic in the Amazon Basin (12-15,21-28) (Figures 1 and 2).

Spellerberg and Hardes (29) describe themajor threats to rain forest conservation asshifting agriculture, cattle ranching, logging, andindustrialization (mining, hydroelectric dams),to which we add land colonization. We

Table 1. Reservoir hosts, triatomines and palm treesparticipating in the life cycle of transmission ofTrypanosoma cruzi in the Amazon BasinMammal hosts of T. cruzia

Primata Marsupialia Edentata Rodentia Carnivora Artiodactyla ChiropteraTriatomines Belminus herreri Cavernicola lenti C. pilosab

Eratyrus mucronatusb

Microtriatoma trinidadensisb

Panstrongylus geniculatusb

P. rufotuberculatusb

P. lignariusb

P. arthruri Rhodnius brethesi R. nasutus R. neglectusb

R. paraensisb

R. pictipesb

R. prolixusb

R. robustusb

Triatoma maculata T. rubrofasciata T. rubrovariaPalm trees Acrocomia aculeata A. sclerocarpac

Astrocaryum aculeatum Attalaea phaleratac

A. vulgare Bactris gasipaes Euterpe oleraceac

E. precatoria Leopoldina piassaba Mauritia flexuosac

Maximiliana elegansc

M. regia Oenocarpus bacaba O. bataua O. mapora Phytelephas macrocarpa Scheelea martiana Sheelea sp.ca>100 mammal wildlife species are reservoirs of T. cruzi (11).bTriatomine species found with T. cruzi infection (12-14).cPalm species with triatomines infected with T. cruzi (13,15,16).

Figure 1. Human cases of acute human Trypanosomacruzi infections in the Amazon Basin (19-28). FrenchGuiana, 15; Colombia, 100; Ecuador, 14; Peru, 85; andBrazilian States: Amapá, 27; Acre, 7; Amazonas, 33;Pará, 57; and Maranhão, 50 cases. Insert shows Paçodo Lumiar county in the island São Luis, State ofMaranhão, an ecoregion vulnerable to humanpredation, where acute T. cruzi infections have beenidentified.


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county is part of the Tocantins moist forestecoregion (1). We worked in 15 villages,separated by partly deforested argilaceouspathways with scattered houses, where mud-walled, thatch-roofed houses are usually locatedbeneath or beside large palm trees. Thesurroundings consist of shady, partly deforestedareas, where dogs, cats, chickens, pigs, cows, andhorses live; no clear delineation separatesperidomestic areas from the dense rain foresthabitat of wild animals. The county’s economydepends on subsistence agriculture and fishing.Raising domestic animals, producing manioc rootflour and grains, and harvesting greens andfruits necessitate clearing areas of forest.

We conducted a serologic survey to assess theprevalence of T. cruzi infection in 25,451 countyresidents >1 and <75 years of age (72% were <30years of age) in these 15 villages with <1,200houses. Fingerprick blood samples from studyparticipants were collected onto Whatman (Clifton,NJ) 1-mm filter paper for seropositivity assess-ment. After air-drying at room temperature, eachset of 10 blood samples was sealed in clean plasticwrap and kept dry in an ice box during the day ofcollection. The blood samples were then storedfrozen until analysis. At the laboratory, filter paperblood samples were punched out and eluted in 100µL of phosphate-buffered saline (PBS), pH 7.4, asdescribed (30). The test was standardized forobtaining 5 µL of blood in 1 cm2 of the filter paper,and serum proteins were eluted in 100 µL of PBS,pH 7.4, yielded a 1:20 final dilution for screeningseropositivity. For quality control, 10% of thesamples were analyzed by a second examiner.

Trapping TriatominesThe strategy for trapping sylvatic triatomine

bugs derived from published work (12,17), as wellas observations by local residents that triatominesattracted by light fly from palm trees to houses atnight. A night visit to one house resulted incapturing two triatomines on the wall near a lightbulb; both these specimens had protozoanflagellates in the intestinal contents.

Researchers and field workers spoke toresidents at clubs or social organizations. Driedtriatomines were displayed, and communityleaders requested that triatomines in houses becaptured and stored (in a 5x3-cm translucentplastic container with holes in the cap). Thisstrategy proved efficient for collecting triatominesin the rainy season, when they invaded the houses.

hypothesized that certain effects of humaneconomic activity (population growth, land use,deforestation, lack of environmental educationprograms, and human predation on mammalsand birds) in a defined ecoregion of this majorecosystem may therefore pose a risk for outbreaksof acute Chagas disease. We evaluated these riskfactors in the Tocantins River moist forestecoregion (1), which is considered vulnerable tohuman intervention. We conducted a pilot studyto assess seropositivity for T. cruzi infections inthe human population of Paço do Lumiar County,state of Maranhão, Brazil, and to search fortriatomines that harbor these infections in palmtrees. We also gathered and analyzed demo-graphic, environmental, and socioeconomic data.

MethodsThe field study was carried out in Paço do

Lumiar county (population 55,000), 20 km fromSão Luiz, capital of the state of Maranhão. The

Figure 2. Human population growth and acute T. cruziinfections in the Brazilian Amazon Basin (19-28). InA, population density increased 2.5-fold in the lastthree decades; in B, acute T. cruzi infections increasedseveral-fold in the same timespan. = break in scale.


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We systematically dissected 23 palm trees(A. phalerata) in backyards in five villages of thecounty. Each tree was cut into segments: stipe,crown shaft, fronds, petiole, and leaves. Eachsegment was carefully searched for insects,mammals, bird nests, and animal vestiges.

During microhabitat dissection, we captured67 nymphs and 95 adults of three species oftriatomines. The precipitin test was used to typeblood in the intestinal contents of 44 adult maleand female triatomines. The test consisted oftwo-dimensional immunodiffusion of blood in theinsects’ intestinal fluid against taxon-specificantisera (31,32).

Trapping and Identifying Wild AnimalsThe rich bird fauna in the research area

included Aratinga jandaia, Buzeo magnirostris,Colombina passerine, Coragyps atratus,Crotophaga ani, Guira guira, Otus choliba,Pitangus sulphuratus, Turdus fumigatus, andTyranus muscivora. We did not capture birdsbecause they are refractory to T. cruzi infections.However, we captured sylvatic mammals (Didel-phis marsupialis) near houses in the study area.These ancient marsupials eat palm-tree fruitsand rest and nest in the clefts between the stipeand their fronds. They leave these hiding placesat night to search for fruits, chicken eggs, babychicks, and food scraps. Using nylon net orwooden box-traps baited with mango andbanana, we captured 12 adult D. marsupialis butwere unable to trap Caluromys sp. seen in thepalm trees around the study area.

Biologic Characterization ofKinetoplastid Flagellates

Parasitic protozoa in the feces of insectvectors of Chagas disease infections or in bloodagar broth were demonstrated directly by lightmicroscopy. Flagellate protozoan infections inD. marsupialis were detected by xenodiagnosis orhemoculture (33). For xenodiagnosis, 20 first-instar uninfected nymphs of Dipetalogastermaximus took a blood meal from each adultD. marsupialis captured in the field. Thirty dayslater, the feces of the triatomines were examinedby microscopy for flagellates. Any parasiticflagellates in the feces of triatomines or inhemocultures were subjected to passage inweanling mice. This procedure consisted ofintraperitoneal injection of a saline dilution of themetacyclic flagellates into mice. Two weeks later,

trypomastigote forms of the parasite wereidentified in the blood of the mice, then 100 µL ofinfected blood was seeded in blood-agar slants,the supernatant of which yielded parasitic forms,which were used for mass production in nutrient-rich liver infusion tryptose medium. One isolatefrom R. pictipes (Rp1) and three isolates fromD. marsupialis (Dm1, Dm2, and Dm3) werecharacterized.

Phenotypic and Genotypic Characterizationof T. cruzi-like Isolates

Specific antibodies in sera from Chagasdisease patients with parasitologically confirmedT. cruzi infection were used as phenotypicmarkers for the counterpart herein calledT. cruzi-like parasitic infection. Binding ofantibodies to epimastigote forms grown in liverinfusion tryptose (LIT) medium and to amastigoteforms in sections of murine tissues was detectedby indirect immunofluorescence assay (30,34).Epimastigote and amastigote forms of thearchetype Berenice stock of T. cruzi were used aspositive controls. For negative controls, parasiticforms from both sources were treated with sera ofT. cruzi antibody-negative persons.

We extracted DNA of parasitic forms fromeach of three flagellate protozoa derived fromD. marsupialis and one isolate of R. pictipes. Theepimastigote forms grown in LIT were used forextraction of nuclear and kinetoplast DNA,essentially as described (35). DNA samples wereanalyzed by polymerase chain reaction (PCR)with specific primers for the constant regions ofminicircles of kDNA and for highly repetitivesequences of nuclear DNA of T. cruzi, asdescribed (35-37). In addition, we used the rDNAnested set of primers D71/72A, which can amplifysequences of 125 and 110 base pairs (bp),respectively, from type II or I parasites (38-41).The reactions were run in parallel with 100 pg ofprotozoan flagellates Rp1, Dm1, Dm2, and Dm3.As positive controls, we used 100 pg of DNAfrom T. cruzi Berenice (Type 1) and Dm28 (Type2). Negative controls were 100 pg of DNA fromLeishmania braziliensis (42) and T. rangeli(43,44).

Formalin-killed epimastigote forms ofT. cruzi-like flagellates from Rp1, Dm1, Dm2,Dm3, and Dm4 and the Berenice stock of T. cruziwere used. The probes consisted of a nuclearDNA sequence PCR amplified with primer setsTcZ1/2 (37). The probe was labeled with biotin


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according to the manufacturer’s protocol. Cellsfixed in glass slides were hybridized with selectedDNA probes and stained with fluorescein-labeledstreptavidin (Sigma Chemical Co., St. Louis, MO).

Each of 10 D. marsupialis trapped in the wildand BALB/c mice receiving T. cruzi-like parasitesisolated from triatomines were subjected tohistopathologic study. The animals were eutha-nized, and tissue samples from organs andtissues were fixed in 10% formalin. Threerepresentative sections of skeletal muscles,heart, esophagus, small and large intestine, liver,kidney, spleen, and lung were stained withhematoxylin and eosin for examination bymicroscopy. An average of six sections from thescent glands of the marsupials was taken forhistopathologic study.

ResultsBy indirect immunofluorescence test for anti-

T. cruzi antibodies in human blood collected onfilter paper (30), 212 (0.83%) of persons testedhad specific antibodies for T. cruzi infections(Figure 3). Positive serologic results in youngpopulations indicate recent transmission andacute infection. Forty-six children <10 years ofage (0.18% of the total study population) wereantibody-positive for T. cruzi and were consid-ered acutely infected.

Our results, which show seroprevalence ofT. cruzi infections in the absence of hematopha-gous bugs or their vestiges (excreta and moltedskins) in houses, prompted us to search fortriatomines in the ecosystem where thepopulation was infected or continues to be at risk.The strategy for capture of triatominesconsisted in surveillance of houses by residents(household members captured bugs in thehouse and placed them in plastic containers) orin dissection of palm trees in backyards ofhouses (16,45-49). This householder-assistedsurveillance and capture method yielded 52triatomine bugs (36 R. pictipes and 16R. neglectus). Triatomine excreta and moltedskins in these houses were neither reported byinhabitants nor detected by field workers.Adult triatomines were captured in houses onlyduring the rainy season. We also captured 133triatomines in 23 palm trees cut down inbackyards in five villages. Careful dissection ofthese trees allowed detection of differentdevelopmental stages of triatomines in clefts ofpalm frond-sheets (Figure 4, Tables 2-4).

Figure 3. Human population growth and acuteTrypanosoma cruzi infections in the Amazoniancounty Paço do Lumiar (19-28). In A, populationdensity increased approximately fourfold in the lastthree decades. In B, autochtonous acute T. cruziinfections increased fourfold in the same timespan(comparing percentages of seropositivity in 41- to 50-year-olds vs. 11- to 20-year-olds), affecting youngerage groups.

Figure 4. Palm tree (Attalaea phalerata) frond-sheetmicrosystem and cycle of transmission of Trypano-soma cruzi. This natural dwelling of triatominespecies and top predators (Didelphis marsupialis,circle) consists of clefts formed by the insertion of frondsheets into the stipe of babassu palm.


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Remains of animal species (e.g., nests, hair,feathers) on which triatomine bugs prey wereidentified in palm trees in backyards in fivevillages. Twice when a tree was cut down, adultopossums (Didelphis, Figure 4) ran out of thefronds into the forest. Nests of marsupials andbirds were easily detected on dissection of palmfronds and crowns. In addition to opossums andbirds, we identified molds and captured andidentified different species of various taxa of

invertebrate and vertebrate animals (50-60) inthe 23 palm trees (Table 5, Figure 5). Molds werefound in stipes, fronds, and crowns, and insects inroots, stipes, inflorescence, fruits, fronds,crowns, and leaves. The clefts formed by frondsheets were particularly rich in Amphibia,Arachnida, and Hemiptera. Triatomines weredetected at the bottom of clefts where marsupialsbuilt their nests. Bird nests were found in thefronds and crowns where abundant species ofinsects were available for predation.

Scarcity of blood flagellates in marsupialsprecluded detection by direct microscopy.However, the metacyclic flagellates recovered byxenodiagnosis were subinoculated in weanlingmice. Two weeks after injection, trypomastigoteforms of the parasitic protozoan morphologicallyindistinguishable from T. cruzi were detected inblood of the mice. To define and furthercharacterize these isolates, we used phenotypicand genotypic molecular characterizations. Inthe first group, antibodies in sera of chronicChagas disease patients reacted indistinctly withantigenic determinants in the surface of T. cruziBerenice and with isolates Dm1, Dm2, and Dm3from D. marsupialis and with Rp1 fromR. pictipes.

Genotypic kDNA and nuclear DNA (nDNA)markers were used to genetically characterizethese wild flagellate protozoan isolates. PCRamplification of template DNA from each of theseT. cruzi isolates showed that kDNA primers S35/36 (35-37) amplified T. cruzi, a laboratorystandard for virulent T. cruzi. In addition, whenwe used PCR with mini-exon intergenic spacerprimers TC/TC1/TC2 (38) and rDNA primersD71/72 (39-41), amplification resulted in thesame bands, using template DNA from wild T.cruzi Dm 28c, Dm1, Dm2, Dm3, and Rp1 and fromBerenice T. cruzi isolated from a patient withacute Chagas disease (Figure 6). These molecularfeatures allowed classification of wild isolates of

Table 4. Type of blood in triatomine bugs captured in palm trees and houses

Bird/ Didelphis/ Canine/Bird Didelphis Didelphis Rodent Rodent Human Equine

Triatomine Total (%) (%) (%) (%) (%) (%) (%)Rhodnius pictipes 28 26.6 26.6 26.6 13.4 6.8 6.8 -R. neglectus 10 40 - 40 - - 20Panstrongylus lignarius 6 100 - - - - -% 36.6 18.2 27.3 9.1 4.5 4.5 4.5*The blood type in the gut of triatomine bugs was identified by the agarose gel precipitin test with taxon-specific antiserum(31,32).

Table 2. Triatomine bugs infected with Trypanosomacruzi in palm trees and houses, Paço do Lumiar,Maranhão, Brazil*

Pan-Rhodnius R. strongylus Total

Triatomines pictipes neglectus lignarius (%)Palm trees

No. captured 89 33 11 133% infected 68 39 27 57

HousesNo. captured 36 16 - 52% infected 28 31 - 41*The triatomines nymphs and adults were captured either bycareful dissection of palm trees or surveillance of houses (12).Flagellate forms in the feces of the Triatomines were detectedby microscopy and further identified as T. cruzi (see text).

Table 3. Developmental stages of Rhodnius pictipes,R. neglectus, and Panstrongylus lignarius found inhouses and palm trees Attalaea phalerata*

Reduviidspecies R. pictipes R. neglectus P. lignarius

Nymphs1st instar 4 3 12nd instar 14 - -3rd instar 9 5 14th instar 6 3 15th instar 14 4 2

AdultsMale 27 10 3Female 29 13 3Total 103 38 11*No nymphs were found in houses.


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Table 5. Trophic network in randomly selected palm trees of an Amazonian county*Network Trophic level† Palm tree localization Local given namesMetaphyta: Attalaea phalerata 1 Rainforest BabaçuMold: (50, 51) Meliolaceae 2 Stipe & crown shaft Bolor Meliola acristaeCatabotrydaceae Catobotrys decidiumMetazoa:Insecta (52, 53) ColeopteraCurculionidae Homalinotus coriaceus Rhynchophorus palmarum R. barbirosstris Amerrhynus inca 3 Stipe, fronds & crown BesouroChrysomelidae 3 Crown Barata do coqueiro Coraliomela brunea Mecistomela marginataScarabaeidae 3 Roots Rola bosta, broca raiz Strategus aloeusBruchidae 3 fronds Broca, bicho do côco Pachimerus nucleorum HomopteraAsphidae 3 Crown Pulgão Cerataphis lataniaeDiaspididae 3 Crown Cochonilha coqueiro Aspidiotus destructor LepidopteraBrassolidae 3 Crown Borboleta Brasssolis sophorae Brassopholis astyra astyra HymenopteraFormicidae Acromyrmex histrix A. landolti A. laticeps migrosetosus 2 & 3 Roots, stipe & fronds Formiga A. nobilis A. lundi carli HemipteraReduvidae (6, 7, 10, 17) 4 fronds & crown Barbeiro Rhodnius pictipes R. neglectus Panstrongylus lignarusAracnidea, Araneae (38) 4Theraphosidae fronds AranhaAmphibia 4 fronds Perereca Scinax spAves (56) 5 fronds & crown Aratinga jandaia Jandaia Buzeo magnirostris Gavião Colombina passerine Rolinha Coragyps atratus Urubú Crotophaga ani Anu Guira-guira Anu branco Otus choliba Caburé Pizangus sulphuratus Bem-te-ví Turdus fumigatus Sabiá Tyranus muscivora TesourinhaMarsupialia (54-59) 5 fronds Didelphis marsupialis Mucura Caluromys sp. Cuíca*Palm trees were randomly selected from five different localities (backyards) in the Paço do Lumiar county.†Trophic levels in microhabitat following the flux of energy: 1) Palm tree, predated by molds, insecta, birds and mammals; 2)molds, predated by Formicinae and Ponarinae; 3) Insects, predated by arachnids, amphibians and birds; (4) Arachnids andamphibians, predated by birds; 5) Birds and mammals, predated by hematophagus insects and man (50-60).


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T. cruzi as phylogenetic type I, while Berenicewas T. cruzi II (11,38-41). These results werefurther confirmed by in situ hybridization of wildT. cruzi with a biotinilated 198-bp sequencederived from Berenice template DNA (Figure 7),which was amplified with specific nDNA primersTcz1/2 (37).

ConclusionsSeveral authors (7,8,17-19,12) described T. cruzi-like flagellate protozoans (instead of T. cruzi) inthe blood of various classes of mammals and inthe feces of triatomine species from the AmazonBasin. We detected blood flagellates in 9 of 10Didelphidae captured in backyards of houses inPaço do Lumiar county. In addition, we isolatedflagellates from feces of R. pictipes, which showedthe same morphologic features described forT. cruzi. In biological characterizations, these

flagellates induced low-level parasitemia inlaboratory mice. However, histopathologic le-sions in marsupials and laboratory mice weresimilar to those described in Chagas diseasepatients. Furthermore, nuclear DNA markersdisplayed all features of the T. cruzi standardsBerenice and Dm 28c T. cruzi, which differedfrom those shown by T. rangeli and L. braziliensis.These data confirm the parasitic flagellatespresent in triatomines and mammals in thisecoregion as T. cruzi.

Birds are refractory to T. cruzi infections (33),but several authors (54-60) describe marsupialsas important wildlife reservoirs of this parasiticprotozoon. We used traps with fruit baits tocapture 12 marsupials, which underwent karyo-typing and parasitologic and pathologic examina-tions. The karyotyping confirmed these mam-mals as D. marsupialis. Nine of 10 trappedmarsupials had protozoan blood flagellatesisolated by xenodiagnosis or hemoculture.Histopathologic study of heart sections from

Figure 5. The informal economy: man carrying ababassu palm frond to be sold as soft thatch. A singlepalm tree enclosing five trophic levels associated withthe Rhodnius pictipes (Hemiptera: Reduviidae)transmission of T. cruzi in the babassu microsystem.

Figure 6. Genotypic characterization of wild-typeflagellates by PCR amplification with rDNA and mini-exon specific primers derived from Trypanosoma cruzi.A, template DNAs amplified with mini-exon intergenicspacer primers (38): Blank, negative control; Tcb,archetypic type II T. cruzi Berenice; Rp1, Dm1, Dm2,and Dm3, flagellates isolated from Rhodnius pictipesand from Didelphis marsupialis; Dm28, standard typeI, sylvatic T. cruzi isolate. B, same template DNAsamplified with rDNA primers (39-41). Tcb yieldedtypical 300-bp band of type II lineage, whereas Rp1,Dm1, Dm2, and Dm3 and Dm28 yielded a 350-bp bandof type I T. cruzi lineage, with mini-exon spacerprimers. In addition, Tcb yielded a typical 125-bp bandof type II, whereas the sylvatic T. cruzi isolates yieldeda 110-bp band of type I, with rDNA primers. Thesefindings confirm sylvatic Rp1, Dm1, Dm2, and Dm3 asT. cruzi.


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these infected animals showed typical myocardi-tis, characterized by mononuclear cell infiltratesand target cell lysis. Inflammatory infiltrateswere seen in skeletal muscles, esophagus, andsmall and large intestines. Histopathologic studyof representative tissue sections taken from theonly opossum that did not have the parasitedetected showed no histopathologic alterations(data not shown).

American trypanosomiasis has been consid-ered an ancient zoonosis in which insect vectorsand mammal hosts sympatrically occupy vastareas of South America (33). Wild T. cruziinfections and the bug vectors are syntopicallyadapted to mammalian host habitats undernatural equilibrium. T. cruzi infections and humanChagas disease occur over a large geographicarea, limited by parallels 42° N to 42°S (33). TheTocantins moist forest, much of which is subjectto severe disturbance of the environmentalequilibrium, provided conditions for diseaseoutbreaks (61-64). The growing human popula-tions encroaching on that natural ecoregion arefed upon by the triatomine vector R. pictipes, andcases of acute T. cruzi infections in humans haveexponentially increased in the last three decades.

In this study, we describe a trophic networkof five levels comprising different speciesdwelling in palm tree microhabitats. A singleclass of top predator mammal (Didelphidae) wasfound in the study area. The absence of other taxaof top wild predators upon which bugs feed maycontribute to peridomiciliar and domiciliar

invasion during the wet season. This observationcontrasts with earlier descriptions of sevenfamilies of mammals, belonging to Primates,Edentates, Marsupials, Carnivores, Rodents,and Chiroptera classes (10,55,57), which werehosts for triatomines in relatively undisturbedecoregions. Elimination of a single class ofinvertebrate or vertebrate animals in a trophicnetwork may be a major risk factor leading tomore triatomine species entering houses andinitiating a new cycle of transmission of T. cruziinfection.

In our study, a child <10 years of age with apositive immunofluorescence test (see methods)was considered a host of acute T. cruzi infection.Considering the age-specific prevalence of T.cruzi infections in adults (30) and the fact that foreach acute case that is clinically identified anestimated 20 to 100 others are unrecognized (34),autochthonous human Chagas disease in theAmazon Basin may reach 7,860 to 39,300 cases.The latter figure is consistent with serologicevidence of T. cruzi infection in the BrazilianAmazon region presented in the national reporton Chagas disease (65). The characteristics oftransmission of infections described here do notindicate a need for insecticide spraying in theAmazon region, for the cycle of transmission ofT. cruzi is deeply embedded in a natural trophicnetwork comprising wild animals belonging toseveral classes and trophic levels.

Risk factors associated with the possibility ofemergence of endemic Chagas disease in the

Figure 7. In situ hybridization of Rhodnius pictipes-derived flagellates with an archetypic Berenice Trypanosomacruzi-specific probe. The byotinylated 198-bp sequence amplified with Tcz1/2 nuclear DNA probe (37) wasrevealed with fluorescein-conjugated streptavidin. This genotypic marker, representing 12.5% of total parasiticnuclear DNA, also confirms this sylvatic isolate as T. cruzi.


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Amazon Basin have been described(10,12,17,19,25,54,55,62-64). First, the broadleafmoist rain forest ecosystem may be invaded bytriatomine species (T. infestans andT. rubrofasciata), which are considered complete-ly adapted to human domiciles, or by othertriatomines (P. megistus and T. brasiliensis, T.pseudomaculata, and T. sordida), which can befound in different ecosystems but frequentlyenter and colonize houses (64). Second, severalAmazon Basin triatomine species (R. pictipes,R. prolixus, R. neglectus, R. nausutus,T. vitticeps, T. rubrovaria and others) can adaptto human dwellings, where they could becomeimportant vectors of the T. cruzi infections (63-69). We found no vestiges of triatominecolonization in houses or their surroundings inour study area. We hypothesize that starvingadult R. pictipes and R. neglectus may leave theirnatural shelters at night to feed on human hosts,probably attracted by light in the houses.

Factors associated with triatomines flyingfrom palm tree to houses need to be clarified. Thescarcity of birds and mammals during the wetseason may be an important factor associatedwith anthropic predation and the presence ofT. cruzi-infected insects in houses in the rainyseason. Domiciliation of triatomines may not berequired for an increasing endemicity of Chagasdisease in the Amazon Basin (13,14,19). Forexample, sylvatic Rhodnius brethesi recently bitpeople harvesting palm fibers in Barcelos in thenorthwestern part of the State of Amazonas, 490km upriver from Manaus, leading to an acutecase of Chagas disease (13). Blood samples fromresidents of this locality tested by immunofluo-rescence assay had 12.5% positivity for anti-T. cruzi antibodies (70). These observationssuggest that to prevent transmission of T. cruziinfections to humans in the Amazon Basin, newstrategies are needed, which will not necessarilybe similar to those used in controlling endemicChagas disease in other ecosystems, such as theCerrado and Caatinga ecosystems in Brazil(1,63,66,67,71).

The risk for endemic Chagas disease in theAmazon Basin appears related to deforestationand new population settlements, shifting cultiva-tion, and rapid human colonization of the vectors’natural ecotope in the last three or more decades,as well as to lack of multiple blood sources (hosts)for the vectors in the rainy season. Amazon Basinperiurban and urban areas where acute cases of

Chagas disease have been reported did not showsigns of colonization of houses with triatominebugs. Instead, transmission of sylvatic T. cruzi tohumans has been associated with sylvatic species(R. pictipes, R. neglectus, P. lignarius), which flyfrom palm trees to houses. New prevention andcontrol strategies should take into considerationrisk factors leading to endemicity of the diseasein the Amazon. An entomologic and epidemio-logic system for surveillance of Chagas disease inAmazonia has been suggested (68,69).

Alternatively, the enormous task of control-ling emerging Chagas disease in the AmazonBasin should rely initially on an information,education, and communication program, whichencourages control measures by the householder(e.g., use of screens, bed nets, insecticide-treatedfabrics, and vegetation management) (69-73).Such a program for prevention of contact withtriatomines should be conducted directly incommunities, elementary schools, and churchesand social clubs, reinforced by social marketingand mass media communications. Furtherstudies are also needed for identifying new andintegrated (chemical and nonchemical) strate-gies required for controlling T. cruzi vectors inthe Amazon Basin, which may not necessarily besimilar to those already shown to be partiallyeffective in controlling the domestic vectors ofendemic Chagas disease in other ecosystems inthe Americas.

AcknowledgmentsWe thank the National Laboratory for Taxonomy of

Triatomines, Fundação Instituto Oswaldo Cruz, Rio deJaneiro, and the Chagas Disease Multidisciplinary ResearchLaboratory, University of Brasília, for performing theprecipitin test.

Dr. Teixeira is professor of pathology at the medicalschool of the University of Brasilia. His research inter-ests focus on endemic infections by kinetoplastic flagel-lates and the pathogeneses of Chagas disease and cuta-neous and visceral leishmaniasis.

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